The cotton agroecosystem is one of the most intensely managed, economically and culturally important fiber crops worldwide, including in the United States of America (U.S.), China, India, Pakistan, and Brazil. The composition and configuration of crop species and semi-natural habitat can have significant effects on ecosystem services such as pollination. Here, we investigated the local-scale effect of crop and semi-natural habitat configuration in a large field (>200 ha in size) cotton agroecosystem on the diversity and abundance of native bees. The interfaces sampled included cotton grown next to cotton, sorghum or semi-natural habitat along with a natural habitat comparator. Collections of native bees across interface types revealed 32 species in 13 genera across 3 families. Average species richness metrics ranged between 20.5 and 30.5, with the highest (30.5) at the interface of cotton and semi-natural habitat. The most abundant species was Melissodes tepaneca Cresson (>4000 individuals, ~75% of bees collected) with a higher number of individuals found in all cotton–crop interfaces compared to the cotton interface with semi-natural habitat or natural habitat alone. It was also found that interface type had a significant effect on the native bee communities. Communities of native bees in the cotton–crop interfaces tended to be more consistent in species richness and abundance. While cotton grown next to semi-natural habitat had higher species richness, the number of bees collected varied. These data suggest that native bee communities persist in large-field cotton agroecosystems. Selected species dominate (i.e., M. tepaneca) and thrive in this large-field cotton system where cotton–crop interfaces are key local landscape features. These data have implications for potential pollination benefits to cotton production. The findings also contribute to a discussion regarding the role of large-field commercial cotton growing systems in conserving native bees.